Lipski et al. Journal of Neuroinflammation (2017) 14:136 DOI 10.1186/s12974-017-0915-5
RESEARCH
Open Access
MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis Deborah A. Lipski1,2*† , Rémi Dewispelaere1,3†, Vincent Foucart1,3,4, Laure E. Caspers3, Matthieu Defrance5, Catherine Bruyns1 and François Willermain1,3,4
Abstract Background: Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU. Methods: EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq. Results: Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45−CD11b− non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C− cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C− cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells. Conclusion: Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity. Keywords: Autoimmune eye disorders, Inflammation, Blood-retinal barrier, Antigen presentation, Co-stimulatory molecules, Microglia, Macrophages, Ly6C, Transcriptome, RNA-Seq
* Correspondence: [email protected] † Equal contributors 1 Ophthalmology Group, IRIBHM (Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire), Université Libre de Bruxelles (ULB), Erasme Campus, Building C, Room C6.117, 808 Route de Lennik, 1070 Brussels, Belgium 2 Ophthalmology Department of Erasme Hospital, Université Libre de Bruxelles (ULB), 808 Route de Lennik, 1070 Brussels, Belgium Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Lipski et al. Journal of Neuroinflammation (2017) 14:136
Background The major histocompatibility complex (MHC) is a set of cell surface proteins divided into two major groups respectively known as class I and class II molecules, which play a fundamental role in adaptive immunity. While MHC class I is ubiquitously expressed by almost all cells, MHC class II is mostly expressed by antigenpresenting cells (APCs) such as monocytes, macrophages, and dendritic cells. These cells are involved in external antigen (Ag) processing and antigenic peptide presentation in the context of MHC class II to CD4+ T helper (TH) cells. Full TH cell activation occurs when the peptide-MHC class II complex interacts with the T cell receptor (TCR), in the presence of signals delivered by the interaction of co-stimulatory molecules such as CD40, CD80, and CD86 on the APC and their ligands on T cells. Interestingly, expression of MHC class II is not strictly restricted to immune cells. It has been demonstrated that non-professional APCs are capable of inducible MHC class II expression, Ag presentation, and even effective T cell reactivation [1, 2]. Aberrant expression of MHC class II by non-professional APCs from targeted organs and subsequent presentation of auto-Ags is now considered to be an important mechanism in the pathogenesis of autoimmune disease processes, including those affecting the eye [3, 4]. Experimental autoimmune uveitis (EAU) is a model of organ autoimmunity in the eye. EAU is mediated by activated TH cells, which are believed to be central in the pathogenesis of human non-infectious uveitis as well [5, 6]. Immunization with IRBP in adjuvant context leads to priming of autoreactive T cells in peripheral lymphoid organs and polarization into TH1 and TH17 cells. These activated TH cells then home to the eye, where they induce blood-retinal barrier (BRB) breakdown and subsequent massive recruitment of diverse inflammatory leukocytes from the circulation [7]. It has been shown that while the first activated T cells enter the eye by chance, regardless of their specificity for retinal or non-retinal Ags, only retina-specific T cells induce EAU [8, 9]. This leads to the conclusion that EAU induction requires TH cell restimulation by in situ Ag recognition. However, the main targets in IRBPinduced EAU are the photoreceptors, which are not believed to express MHC class II. In this context, a tremendous series of works have tried to determine which cell types are responsible for intra-ocular Ag presentation during EAU. Both resident retinal cells [10] and infiltrating hematopoietic cells [11] have been proposed, with inconsistent results. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express MHC class II during adoptive transfer (AT) EAU.
Page 2 of 22
Methods Reagents and animals
Interphotoreceptor retinoid-binding peptide (IRBP) 1–20 (GPTHLFQPSLVLDMAKVLLD), representing residues 1–20 of human IRBP, was synthesized by New England Peptide (Gardner, MA, USA). Pertussis toxin (PTX) and complete Freund’s adjuvant (CFA) were purchased from Sigma-Aldrich (Bornem, Belgium). Pathogen-free female C57BL/6 mice (6 to 10 weeks old), purchased from Janvier (Genest St Isle, France) were housed at the animal facilities in accordance with the European guidelines. Animal treatment conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All cells were cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 10% fetal bovine serum, 1% L-glutamine, 1% sodium-pyruvate, 100 IU/ml penicillin, 100 g/ml streptomycin, 5.10−5 M β-mercaptoethanol in a 5% CO2, and 95% humidity incubator. Classical and adoptive transfer models of experimental autoimmune uveitis
Classical EAU was induced by immunization of naive C57BL/6 mice with a subcutaneous injection in each hind leg of 50 μl of a mixture containing 500 μg/100 μl IRBP peptide 1–20 in a 1:1 emulsion with CFA enriched with 2.5 mg/ml of heat-inactivated mycobacterium tuberculosis. All animals simultaneously received an intraperitoneal injection of 1 μg of PTX. Adoptive transfer EAU was induced by AT of autoreactive lymphocytes following the protocol of Shao H et al. [12]. Briefly, animals were immunized as mentioned above. Twelve days after immunization, mice were euthanized and their spleen and draining lymph nodes dissected and dissociated. Spleen cell suspensions were enriched in T lymphocytes through passage on nylon wool fiber columns, then pooled with total lymph node cells and restimulated in vitro with IRBP1-20 (1 μg/ml). After 2 days in culture, cells were injected intraperitoneally into naive C57BL/6 mice (5 × 106 cells/mouse). Disease grading
A clinical grading was performed at day 14 or day 21 after disease induction. Mice were anesthetized by a 50μl intraperitoneal injection of a Rompun (0.2%) and Ketalar (20 mg/ml) mixture. The pupils were dilated with tropicamid (5 mg/ml) and phenylephrine (1.5 mg/ml), and the eyes were examined under the slit-lamp of a surgical microscope (Zeiss, Göttingen, Germany) by using a cover slip coated in a viscoelastic gel (synthetic polymer of acrylic acid 2 mg/g, Vidisic, Tramedico, Belgium) and positioned on the cornea. The clinical grading was performed independently by two ophthalmologists, based on a system adapted from Xu et al. [13]. Briefly, vitritis, optic neuropathy, retinitis, and
Lipski et al. Journal of Neuroinflammation (2017) 14:136
vasculitis were separately scored in each eye, from 0 (no disease) to 4 (highly severe disease) with half-point increments and averaged to generate the clinical score of the eye on a scale from 1 to 4. The clinical score attributed to one mouse corresponds to the mean of the scores of the two eyes. Immunohistology Immunofluorescence stainings on retinal cryosections
At day 14 or 21 after disease induction, the eyes were collected, prefixed for 6 h at 4 °C in PFA (paraformaldehyde) 4% and sucrose 3%, and then put in three successive baths containing 5, 10, and 18% sucrose in PBS, respectively, for 24 h each. The entire eyes were embedded in OCT medium (Sakura, Antwerp, Belgium) and cut in 16-μm-thick frozen sections using a cryostat (CM3050S Leica). The MOM (mouse-on-mouse) Basic Kit (Vector Laboratories, Labconsult, Brussels) was used to prevent high background staining. Cryosections were fixed with PFA 4% for 15 min and blocked in TBS (Tris 10 mM, NaCl 0.9%, pH 7.6) supplemented with MOM IgG blocking solution and Triton 0.3% for 2 h. Sections were incubated overnight with the following primary antibodies, alone or in different combinations as indicated in the results: anti-MHC class II (rat, 1/200; BD Biosciences, Erembodegem, Belgium), anti-GFAP (mouse, 1/500; Millipore, Brussels, Belgium), anti-endoglin (goat, 3/ 500; BD Biosciences), anti-CD31 (goat, 1/200, R&D systems, Abingdon), and anti-IBA1 (goat, 1/100, Abcam, Cambridge, UK), diluted in TBS supplemented with MOM kit protein concentrate. After three washings in TBS, the sections were incubated in the dark for 1 h30 with species-specific secondary antibodies coupled to different fluorochromes, as indicated in data, then with Hoechst to stain the nuclei (Invitrogen, Gent, Belgium). After several washings, the sections were mounted in Glycergel (Dako, Agilent Technologies, Diegem, Belgium) supplemented with 2.5% Dabco (Sigma-Aldrich). Pictures of immunostainings were acquired using an AxioImager Z1 microscope equipped with an AxioCamMR camera (Carl Zeiss, Inc.) using the z-stack mode of the Axiovision acquisition software. Z-stacks were processed using the Imaris deconvolution software. Differential interference contrast (DIC) images were recorded on an AxioImager Z1 (Zeiss) upright widefield microscope using a Plan Apochromat 20x\0.8 NA (Zeiss) air objective combined with the corresponding EC PN (II) prism slider (Zeiss) under the objective and polarizing filters in the light path. Immunofluorescence stainings on retinal wholemount preparations
At day 21 after disease induction, the eyes were collected and immediately immersed in PFA 4% for 1 h at 4 °C. The eyes were then dissected in ice-cold PBS: the
Page 3 of 22
anterior segment of the globe, crystalline lens, and vitreous were removed and the retina was carefully peeled from the retinal pigment epithelium. Whole retinas were fixed in 70% ethanol for 1 h, rinsed three times in PBS (10 min each), blocked with a solution containing 3% milk and 3% bovine serum albumin in PBS for 1 h and incubated with anti-MHC class II (rat, 1/200; BD Biosciences, Erembodegem, Belgium) and anti-endoglin (goat, 1/200; BD Biosciences) antibodies in a PBS-BSA1% solution overnight at room temperature. The retinas were rinsed three times in PBS (20 min each) and incubated sequentially for 1 h with each secondary antibody. After three final washings in PBS (20 min each), each retina was flattened by radial incisions and mounted with Vectashield antifade mounting medium with DAPI (Labconsult, Brussels). Preparation of retinal single-cell suspensions
Mice were sacrificed at day 14 or 21 after disease induction, and the eyes immediately enucleated. Eyes were carefully hemisected in HBSS buffer containing penicillin/streptomycin 1%, with surgical scissors under a surgical microscope. Retinal tissue was isolated and rinsed in HBSS medium. The two retinas of each mouse were pooled and cut into small pieces before enzymatic digestion in 3 ml HBSS containing 1.6 mg/ml Liberase (Roche, Vilvoorde, Belgium) and 0.1 mg/ml DNase I (Sigma-Aldrich) at 37 °C for 45 min. Cell dissociation was stimulated by pipetting every 15 min. Cells were washed with DMEM/10% FBS and filtered through a 40-μm cell strainer to obtain a single-cell suspension [14]. The yield was approximately 2 to 3 million retinal cells per mouse. Fluorescence-activated cell sorting (FACS) analysis
Retinal single-cell suspensions were tested by flow cytometry (FC) for their membrane expression of MHC class II (I-A/I-E), CD45 (pan-leucocyte marker), CD11b (myeloid cell marker), Ly6C (monocyte/macrophage marker), CD31 (endothelial cell marker), CD40, CD80 and CD86 (co-stimulatory molecules), and F4/80 (panmacrophage marker) using specific antibodies (BD Biosciences) coupled to different fluorochromes. For Lipocalin 2 (Lcn2) and Cysteinyl Leukotriene Receptor 1 (Cysltr1), unconjugated primary antibodies (from R&D systems and Abcam, respectively) and species-specific secondary antibodies coupled to Alexa488 were used. Cells were incubated with the relevant antibodies for 20 min at 4 °C, washed and resuspended in FACS buffer. For Lcn2 intracytoplasmic staining, cells were first incubated with membrane antibodies then permeabilized with Cytofix/cytoperm fixation/permeabilization solution (BD Biosciences) before incubation with the anti-Lcn2 antibody. Live cells were gated with Hoechst 1/4000 and
Lipski et al. Journal of Neuroinflammation (2017) 14:136
debris and doublets were excluded (the complete gating strategy is illustrated in Additional file 1: Figure S1). Up to one million total cells per sample were analyzed on a LSR-Fortessa flow cytometer using the CellQuest Software (BD Biosciences). Isotypes and Fluorescence minus one (FMO) controls were used for accurate gating. Compensations were performed using BD CompBeads (BD Biosciences). Analysis of retinal cell gene expression Purification of different MHC class II+ populations
Three weeks after AT, mice were sacrificed and retinal single-cell suspensions prepared as described above. Cells were stained with PE-labeled anti-MHC class II, PECy7-labeled anti-CD45, FITC-labeled anti-CD11b and APC-labeled anti-Ly6C antibodies. MHC class II+CD45 + CD11b+Ly6C+ cells (referred to as Plus), MHC class II + CD45+CD11b+Ly6C− cells (referred to as Minus), and MHC class II+CD45−CD11b−Ly6C− (referred to as nonhematopoietic or NH) were separately sorted by preparative FC using a FACSAria with the FACSDiva Software (BD). Due to the low cell number obtained from each mouse (around 1000 Plus cells), three mice were pooled
Page 4 of 22
to generate each sample. Cells were sorted directly in lysis buffer, vortexed for 30 s and flash frozen in liquid nitrogen. The purity of the sorted cell populations was evaluated by FC re-analysis of sorted cells (Additional file 2: Figure S2). RNA extraction
RNA extraction was performed using the MiRNeasy MicroKit (Qiagen) according to the manufacturer’s recommendations and a DNase step to avoid DNA contamination. RNA quality was assessed using the Agilent 2100 Bioanalyzer with RNA 6000 Pico kit (Agilent Technologies). RNA processing and RNA sequencing
Indexed cDNA libraries were prepared using the Ovation Single Cell RNAseq system (Nugen). The multiplexed libraries were loaded and sequences were produced using a TruSeq PE cluster and SBS-kit on a HiSeq 1500 (Illumina). Approximately 25 million paired-end reads/sample were mapped against the mouse reference genome (NCBI Build 37/UCSC mm9) using STAR software to generate read alignments for
a
b
Fig. 1 MHC class II retinal expression is highly induced during EAU. Three weeks after adoptive transfer, eye cryosections were prepared and stained for MHC class II (green) and IBA1 (red) detection. Naive eyes were used as control. Cell nuclei were stained with Hoechst (blue). Each picture was chosen as representative of an experiment conducted on three or more animals. a Naive retina. b Adoptive transfer EAU retina (grade 3.5)
Lipski et al. Journal of Neuroinflammation (2017) 14:136
each sample. Expression levels were quantified using the featureCounts [15] tool and the UCSC RefSeq gene annotation as a reference (exons only, genes as meta features). Differential analysis between the groups was performed using the EdgeR package (quasi-likelihood F-tests). Normalized expression levels were estimated using the EdgeR rpm function and converted to log2 FPKM (fragments per kilobase of exon per million mapped reads) after resetting low FPKMs to 1. To perform blind clustering analysis, genes were selected based
Page 5 of 22
on the overall variance between samples (independently of their category), by keeping only the 30 most variant ones. Functional analysis was performed using the DAVID web-based functional annotation tool [16]. Statistical analysis
Statistical analysis was performed using Kruskal-Wallis, ANOVA, Tukey post-hoc multiple comparisons test, and Student’s t test. Only p values
RESEARCH
Open Access
MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis Deborah A. Lipski1,2*† , Rémi Dewispelaere1,3†, Vincent Foucart1,3,4, Laure E. Caspers3, Matthieu Defrance5, Catherine Bruyns1 and François Willermain1,3,4
Abstract Background: Controversy exists regarding which cell types are responsible for autoantigen presentation in the retina during experimental autoimmune uveitis (EAU) development. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express major histocompatibility complex (MHC) class II during EAU. Methods: EAU was induced in C57BL/6 mice by adoptive transfer of autoreactive lymphocytes from IRBP1-20-immunized animals. MHC class II expression was studied by immunostainings on eye cryosections. For flow cytometry (FC) analysis, retinas were dissected and enzymatically digested into single-cell suspensions. Three MHC class II+ retinal cell populations were sorted by FC, and their RNA processed for RNA-Seq. Results: Immunostainings demonstrate strong induction of MHC class II expression in EAU, especially in the inner retina at the level of inflamed vessels, extending to the outer retinal layers and the subretinal space in severely inflamed eyes. Most MHC class II+ cells express the hematopoietic marker IBA1. FC quantitative analyses demonstrate that MHC class II induction significantly correlates with disease severity and is associated with upregulation of co-stimulatory molecule expression. In particular, most MHC class IIhi cells express co-stimulatory molecules during EAU. Further phenotyping identified three MHC class II+ retinal cell populations: CD45−CD11b− non-hematopoietic cells with low MHC class II expression and CD45+CD11b+ hematopoietic cells with higher MHC class II expression, which can be further separated into Ly6C+ and Ly6C− cells, possibly corresponding to infiltrating macrophages and resident microglia. Transcriptome analysis of the three sorted populations leads to a clear sample clustering with some enrichment in macrophage markers and microglial cell markers in Ly6C+ and Ly6C− cells, respectively. Functional annotation analysis reveals that both hematopoietic cell populations are more competent in MHC class II-associated antigen presentation and in T cell activation than non-hematopoietic cells. Conclusion: Our results highlight the potential of cells of hematopoietic origin in local antigen presentation, whatever their Ly6C expression. Our work further provides a first transcriptomic study of MHC class II-expressing retinal cells during EAU and delivers a series of new candidate genes possibly implicated in the pathogenesis of retinal autoimmunity. Keywords: Autoimmune eye disorders, Inflammation, Blood-retinal barrier, Antigen presentation, Co-stimulatory molecules, Microglia, Macrophages, Ly6C, Transcriptome, RNA-Seq
* Correspondence: [email protected] † Equal contributors 1 Ophthalmology Group, IRIBHM (Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire), Université Libre de Bruxelles (ULB), Erasme Campus, Building C, Room C6.117, 808 Route de Lennik, 1070 Brussels, Belgium 2 Ophthalmology Department of Erasme Hospital, Université Libre de Bruxelles (ULB), 808 Route de Lennik, 1070 Brussels, Belgium Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Lipski et al. Journal of Neuroinflammation (2017) 14:136
Background The major histocompatibility complex (MHC) is a set of cell surface proteins divided into two major groups respectively known as class I and class II molecules, which play a fundamental role in adaptive immunity. While MHC class I is ubiquitously expressed by almost all cells, MHC class II is mostly expressed by antigenpresenting cells (APCs) such as monocytes, macrophages, and dendritic cells. These cells are involved in external antigen (Ag) processing and antigenic peptide presentation in the context of MHC class II to CD4+ T helper (TH) cells. Full TH cell activation occurs when the peptide-MHC class II complex interacts with the T cell receptor (TCR), in the presence of signals delivered by the interaction of co-stimulatory molecules such as CD40, CD80, and CD86 on the APC and their ligands on T cells. Interestingly, expression of MHC class II is not strictly restricted to immune cells. It has been demonstrated that non-professional APCs are capable of inducible MHC class II expression, Ag presentation, and even effective T cell reactivation [1, 2]. Aberrant expression of MHC class II by non-professional APCs from targeted organs and subsequent presentation of auto-Ags is now considered to be an important mechanism in the pathogenesis of autoimmune disease processes, including those affecting the eye [3, 4]. Experimental autoimmune uveitis (EAU) is a model of organ autoimmunity in the eye. EAU is mediated by activated TH cells, which are believed to be central in the pathogenesis of human non-infectious uveitis as well [5, 6]. Immunization with IRBP in adjuvant context leads to priming of autoreactive T cells in peripheral lymphoid organs and polarization into TH1 and TH17 cells. These activated TH cells then home to the eye, where they induce blood-retinal barrier (BRB) breakdown and subsequent massive recruitment of diverse inflammatory leukocytes from the circulation [7]. It has been shown that while the first activated T cells enter the eye by chance, regardless of their specificity for retinal or non-retinal Ags, only retina-specific T cells induce EAU [8, 9]. This leads to the conclusion that EAU induction requires TH cell restimulation by in situ Ag recognition. However, the main targets in IRBPinduced EAU are the photoreceptors, which are not believed to express MHC class II. In this context, a tremendous series of works have tried to determine which cell types are responsible for intra-ocular Ag presentation during EAU. Both resident retinal cells [10] and infiltrating hematopoietic cells [11] have been proposed, with inconsistent results. In this study, we aimed to identify and characterize the retinal resident and infiltrating cells susceptible to express MHC class II during adoptive transfer (AT) EAU.
Page 2 of 22
Methods Reagents and animals
Interphotoreceptor retinoid-binding peptide (IRBP) 1–20 (GPTHLFQPSLVLDMAKVLLD), representing residues 1–20 of human IRBP, was synthesized by New England Peptide (Gardner, MA, USA). Pertussis toxin (PTX) and complete Freund’s adjuvant (CFA) were purchased from Sigma-Aldrich (Bornem, Belgium). Pathogen-free female C57BL/6 mice (6 to 10 weeks old), purchased from Janvier (Genest St Isle, France) were housed at the animal facilities in accordance with the European guidelines. Animal treatment conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All cells were cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 10% fetal bovine serum, 1% L-glutamine, 1% sodium-pyruvate, 100 IU/ml penicillin, 100 g/ml streptomycin, 5.10−5 M β-mercaptoethanol in a 5% CO2, and 95% humidity incubator. Classical and adoptive transfer models of experimental autoimmune uveitis
Classical EAU was induced by immunization of naive C57BL/6 mice with a subcutaneous injection in each hind leg of 50 μl of a mixture containing 500 μg/100 μl IRBP peptide 1–20 in a 1:1 emulsion with CFA enriched with 2.5 mg/ml of heat-inactivated mycobacterium tuberculosis. All animals simultaneously received an intraperitoneal injection of 1 μg of PTX. Adoptive transfer EAU was induced by AT of autoreactive lymphocytes following the protocol of Shao H et al. [12]. Briefly, animals were immunized as mentioned above. Twelve days after immunization, mice were euthanized and their spleen and draining lymph nodes dissected and dissociated. Spleen cell suspensions were enriched in T lymphocytes through passage on nylon wool fiber columns, then pooled with total lymph node cells and restimulated in vitro with IRBP1-20 (1 μg/ml). After 2 days in culture, cells were injected intraperitoneally into naive C57BL/6 mice (5 × 106 cells/mouse). Disease grading
A clinical grading was performed at day 14 or day 21 after disease induction. Mice were anesthetized by a 50μl intraperitoneal injection of a Rompun (0.2%) and Ketalar (20 mg/ml) mixture. The pupils were dilated with tropicamid (5 mg/ml) and phenylephrine (1.5 mg/ml), and the eyes were examined under the slit-lamp of a surgical microscope (Zeiss, Göttingen, Germany) by using a cover slip coated in a viscoelastic gel (synthetic polymer of acrylic acid 2 mg/g, Vidisic, Tramedico, Belgium) and positioned on the cornea. The clinical grading was performed independently by two ophthalmologists, based on a system adapted from Xu et al. [13]. Briefly, vitritis, optic neuropathy, retinitis, and
Lipski et al. Journal of Neuroinflammation (2017) 14:136
vasculitis were separately scored in each eye, from 0 (no disease) to 4 (highly severe disease) with half-point increments and averaged to generate the clinical score of the eye on a scale from 1 to 4. The clinical score attributed to one mouse corresponds to the mean of the scores of the two eyes. Immunohistology Immunofluorescence stainings on retinal cryosections
At day 14 or 21 after disease induction, the eyes were collected, prefixed for 6 h at 4 °C in PFA (paraformaldehyde) 4% and sucrose 3%, and then put in three successive baths containing 5, 10, and 18% sucrose in PBS, respectively, for 24 h each. The entire eyes were embedded in OCT medium (Sakura, Antwerp, Belgium) and cut in 16-μm-thick frozen sections using a cryostat (CM3050S Leica). The MOM (mouse-on-mouse) Basic Kit (Vector Laboratories, Labconsult, Brussels) was used to prevent high background staining. Cryosections were fixed with PFA 4% for 15 min and blocked in TBS (Tris 10 mM, NaCl 0.9%, pH 7.6) supplemented with MOM IgG blocking solution and Triton 0.3% for 2 h. Sections were incubated overnight with the following primary antibodies, alone or in different combinations as indicated in the results: anti-MHC class II (rat, 1/200; BD Biosciences, Erembodegem, Belgium), anti-GFAP (mouse, 1/500; Millipore, Brussels, Belgium), anti-endoglin (goat, 3/ 500; BD Biosciences), anti-CD31 (goat, 1/200, R&D systems, Abingdon), and anti-IBA1 (goat, 1/100, Abcam, Cambridge, UK), diluted in TBS supplemented with MOM kit protein concentrate. After three washings in TBS, the sections were incubated in the dark for 1 h30 with species-specific secondary antibodies coupled to different fluorochromes, as indicated in data, then with Hoechst to stain the nuclei (Invitrogen, Gent, Belgium). After several washings, the sections were mounted in Glycergel (Dako, Agilent Technologies, Diegem, Belgium) supplemented with 2.5% Dabco (Sigma-Aldrich). Pictures of immunostainings were acquired using an AxioImager Z1 microscope equipped with an AxioCamMR camera (Carl Zeiss, Inc.) using the z-stack mode of the Axiovision acquisition software. Z-stacks were processed using the Imaris deconvolution software. Differential interference contrast (DIC) images were recorded on an AxioImager Z1 (Zeiss) upright widefield microscope using a Plan Apochromat 20x\0.8 NA (Zeiss) air objective combined with the corresponding EC PN (II) prism slider (Zeiss) under the objective and polarizing filters in the light path. Immunofluorescence stainings on retinal wholemount preparations
At day 21 after disease induction, the eyes were collected and immediately immersed in PFA 4% for 1 h at 4 °C. The eyes were then dissected in ice-cold PBS: the
Page 3 of 22
anterior segment of the globe, crystalline lens, and vitreous were removed and the retina was carefully peeled from the retinal pigment epithelium. Whole retinas were fixed in 70% ethanol for 1 h, rinsed three times in PBS (10 min each), blocked with a solution containing 3% milk and 3% bovine serum albumin in PBS for 1 h and incubated with anti-MHC class II (rat, 1/200; BD Biosciences, Erembodegem, Belgium) and anti-endoglin (goat, 1/200; BD Biosciences) antibodies in a PBS-BSA1% solution overnight at room temperature. The retinas were rinsed three times in PBS (20 min each) and incubated sequentially for 1 h with each secondary antibody. After three final washings in PBS (20 min each), each retina was flattened by radial incisions and mounted with Vectashield antifade mounting medium with DAPI (Labconsult, Brussels). Preparation of retinal single-cell suspensions
Mice were sacrificed at day 14 or 21 after disease induction, and the eyes immediately enucleated. Eyes were carefully hemisected in HBSS buffer containing penicillin/streptomycin 1%, with surgical scissors under a surgical microscope. Retinal tissue was isolated and rinsed in HBSS medium. The two retinas of each mouse were pooled and cut into small pieces before enzymatic digestion in 3 ml HBSS containing 1.6 mg/ml Liberase (Roche, Vilvoorde, Belgium) and 0.1 mg/ml DNase I (Sigma-Aldrich) at 37 °C for 45 min. Cell dissociation was stimulated by pipetting every 15 min. Cells were washed with DMEM/10% FBS and filtered through a 40-μm cell strainer to obtain a single-cell suspension [14]. The yield was approximately 2 to 3 million retinal cells per mouse. Fluorescence-activated cell sorting (FACS) analysis
Retinal single-cell suspensions were tested by flow cytometry (FC) for their membrane expression of MHC class II (I-A/I-E), CD45 (pan-leucocyte marker), CD11b (myeloid cell marker), Ly6C (monocyte/macrophage marker), CD31 (endothelial cell marker), CD40, CD80 and CD86 (co-stimulatory molecules), and F4/80 (panmacrophage marker) using specific antibodies (BD Biosciences) coupled to different fluorochromes. For Lipocalin 2 (Lcn2) and Cysteinyl Leukotriene Receptor 1 (Cysltr1), unconjugated primary antibodies (from R&D systems and Abcam, respectively) and species-specific secondary antibodies coupled to Alexa488 were used. Cells were incubated with the relevant antibodies for 20 min at 4 °C, washed and resuspended in FACS buffer. For Lcn2 intracytoplasmic staining, cells were first incubated with membrane antibodies then permeabilized with Cytofix/cytoperm fixation/permeabilization solution (BD Biosciences) before incubation with the anti-Lcn2 antibody. Live cells were gated with Hoechst 1/4000 and
Lipski et al. Journal of Neuroinflammation (2017) 14:136
debris and doublets were excluded (the complete gating strategy is illustrated in Additional file 1: Figure S1). Up to one million total cells per sample were analyzed on a LSR-Fortessa flow cytometer using the CellQuest Software (BD Biosciences). Isotypes and Fluorescence minus one (FMO) controls were used for accurate gating. Compensations were performed using BD CompBeads (BD Biosciences). Analysis of retinal cell gene expression Purification of different MHC class II+ populations
Three weeks after AT, mice were sacrificed and retinal single-cell suspensions prepared as described above. Cells were stained with PE-labeled anti-MHC class II, PECy7-labeled anti-CD45, FITC-labeled anti-CD11b and APC-labeled anti-Ly6C antibodies. MHC class II+CD45 + CD11b+Ly6C+ cells (referred to as Plus), MHC class II + CD45+CD11b+Ly6C− cells (referred to as Minus), and MHC class II+CD45−CD11b−Ly6C− (referred to as nonhematopoietic or NH) were separately sorted by preparative FC using a FACSAria with the FACSDiva Software (BD). Due to the low cell number obtained from each mouse (around 1000 Plus cells), three mice were pooled
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to generate each sample. Cells were sorted directly in lysis buffer, vortexed for 30 s and flash frozen in liquid nitrogen. The purity of the sorted cell populations was evaluated by FC re-analysis of sorted cells (Additional file 2: Figure S2). RNA extraction
RNA extraction was performed using the MiRNeasy MicroKit (Qiagen) according to the manufacturer’s recommendations and a DNase step to avoid DNA contamination. RNA quality was assessed using the Agilent 2100 Bioanalyzer with RNA 6000 Pico kit (Agilent Technologies). RNA processing and RNA sequencing
Indexed cDNA libraries were prepared using the Ovation Single Cell RNAseq system (Nugen). The multiplexed libraries were loaded and sequences were produced using a TruSeq PE cluster and SBS-kit on a HiSeq 1500 (Illumina). Approximately 25 million paired-end reads/sample were mapped against the mouse reference genome (NCBI Build 37/UCSC mm9) using STAR software to generate read alignments for
a
b
Fig. 1 MHC class II retinal expression is highly induced during EAU. Three weeks after adoptive transfer, eye cryosections were prepared and stained for MHC class II (green) and IBA1 (red) detection. Naive eyes were used as control. Cell nuclei were stained with Hoechst (blue). Each picture was chosen as representative of an experiment conducted on three or more animals. a Naive retina. b Adoptive transfer EAU retina (grade 3.5)
Lipski et al. Journal of Neuroinflammation (2017) 14:136
each sample. Expression levels were quantified using the featureCounts [15] tool and the UCSC RefSeq gene annotation as a reference (exons only, genes as meta features). Differential analysis between the groups was performed using the EdgeR package (quasi-likelihood F-tests). Normalized expression levels were estimated using the EdgeR rpm function and converted to log2 FPKM (fragments per kilobase of exon per million mapped reads) after resetting low FPKMs to 1. To perform blind clustering analysis, genes were selected based
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on the overall variance between samples (independently of their category), by keeping only the 30 most variant ones. Functional analysis was performed using the DAVID web-based functional annotation tool [16]. Statistical analysis
Statistical analysis was performed using Kruskal-Wallis, ANOVA, Tukey post-hoc multiple comparisons test, and Student’s t test. Only p values
